Acinetobacter baumannii (A. baumannii) is a human pathogen responsible for recent increases in nosocomial infections of both military and civilian populations. Further emergence of the pathogen's broad resistance to available antibiotics has emphasized the need for truly novel antibacterial therapeutics. We have previously engineered a naturally occurring bactericidal protein, R-type pyocin, to kill bacterial pathogens beyond the natural killing spectrum of the pyocin and to kill by first binding via its tail fibers to a targeted virulence factor accessible on the pathogen's surface. As a result, pathogens emerging resistant to the engineered pyocin have compromised virulence. Herein we propose to create such bactericidal agents for A. baumannii. R-type pyocins are high molecular weight protein complexes produced by some Pseudomonas aeruginosa strains and which specifically kill other strains of the same species. Because the bactericidal mechanism of R-type pyocins and their engineered derivatives is completely different from the mechanisms of action of traditional antibiotics, R-type pyocins are unaffected by the mechanisms deployed by bacteria to resist antibiotics. Our goal is to generate tail fibers with novel binding phenotypes that can be selected and integrated into R-type pyocins. Diversity Generating Retroelements (DGRs) are a newly discovered means to diversify DNA sequences and the proteins they encode. The DGR provides a means to generate libraries of proteins that are highly variable at precise locations and can be used to select variants with desired physical, chemical or biological properties. In collaboration with Walter Reed Army Institute of Research (WRAIR) we shall isolate genes encoding tail fibers that target surface accessible virulence factors of A. baumannii isolates from wounded warfighters and deploy those genes to create R-type pyocins specifically bactericidal for multiple strains of the pathogen. The engineered R- type pyocins shall be evaluated for their efficacy in vitro and in vivo, the latter in an animal model of wound infection at WRAIR. Mutants of A. baumannii selected for resistance to engineered pyocins that target virulence factors shall be assessed for their pathogenicity in the same animal model. This will allow us to determine whether the resistant A. baumannii organisms have compromised virulence in vivo, as we have previously shown occurs for E. coli O157:H7 mutants resistant to an R-type pyocin that was engineered to target one of its virulence factors. PUBLIC HEALTH RELEVANCE: Acinetobacter baumannii is a species of bacteria readily found in soil and water and has an uncanny ability to resist old and new antibiotics. These bacteria are usually harmless to healthy humans but have found a significant and dangerous niche in hospitalized patients, both civilian and military, where they cause prolonged and serious morbidity in the form of broadly antibiotic-resistant wound and bone infections, pneumonia, and urinary tract infections. We have previously engineered naturally occurring antibacterial protein particles, called R-type pyocins, to redirect their bacterial killing ability towards disease-causing bacteria that they do not kill naturally. We propose to create R-type pyocins that kill A. baumannii specifically. Importantly, R-type pyocins kill bacteria by a mechanism completely different than those mechanisms used by any antibiotic;and therefore, R- type pyocins are not subject to the mechanisms that bacteria use to resist antibiotics. We have previously engineered R-type pyocins to target bacteria by first binding to molecules on the bacterial surface that are required for the bacteria to cause disease ("virulence factors"). To become resistant to the targeted pyocin, bacteria must lose or modify that virulence factor and compromise their ability to cause infections. In collaboration with Walter Reed Army Institute of Research (WRAIR) we expect to engineer R-type pyocins to target virulence factors prevalent on the surfaces of A. baumannii bacteria isolated from wounded soldiers. Also in collaboration with WRAIR we shall evaluate the effectiveness of our engineered pyocins in an animal model of wounds infected by A. baumannii.